Primary Structure Analysis Research Articles

In Vitro Selection and Characterization of a Light-up DNA Aptamer for Thiazole Orange.

A new DNA aptamer that binds to the target Thiazole Orange-biotin (TO1-biotin) was isolated after nine rounds of in vitro selection. The selection was performed on streptavidin-coated beads with the target bound to the surface and with free dye in solution in higher selection rounds to select for slower off-rate binding. Using next-generation sequencing (NGS), the libraries after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. Several sequence families emerged, showing superior fluorescence enhancement and high affinity for the target compared to the other families obtained by NGS analysis. These sequence families were further studied to understand the binding interactions better. Primary sequence and secondary structure analysis tools were used to identify a hypothetical three-tiered G-quadruplex motif for these families. This indicates that the TO1-biotin DNA aptamer identified here uses a similar ligand-binding topology to the original Mango RNA aptamer.

Characterization of two carbonic anhydrase isoforms in the pulmonate snail (Lymnaea Stagnalis) and their involvement in Molluskan calcification

Calcifying organisms are suffering from negative impacts induced by climate change, such as CO2-induced acidification, which may impair external calcified structures. Freshwater mollusks have the potential to suffer more from CO2-induced acidification than marine calcifiers due to the lower buffering capacity of many freshwater systems. One of the most important enzymes contributing to the biomineralization reaction is carbonic anhydrase (CA), which catalyzes the reversible conversion of CO2 to bicarbonate, the major carbon source of the calcareous structure in calcifiers. In this study we characterized two α-CA isoforms (LsCA1 and LsCA4) from the freshwater snail Lymnaea stagnalis using a combination of gene sequencing, gene expression, phylogenetic analysis and biochemical assays. Both CA isoforms demonstrated high expression levels in the mantle tissue, the major site for biomineralization. Furthermore, expression of LsCA4 during development parallels shell formation. The primary protein structure analysis, active site configuration and the catalytic activity of LsCA4 together suggest that the LsCA4 is embedded in the apical and basolateral membranes of mantle cells; while LsCA1 is proposed to be cytosolic and might play an important role in acid-base regulation. These findings of LsCA isoforms form a strong basis for a more detailed physiological understanding of the effects of elevated CO2 on calcification in freshwater mollusks.

Green Hydrogen, a Solution for Replacing Fossil Fuels to Reduce CO2 Emissions

The article examines the role of green hydrogen in reducing CO2 emissions in the transition to climate neutrality, highlighting both its benefits and challenges. It starts by discussing the production of green hydrogen from renewable sources and provides a brief analysis of primary resource structures for energy production in European countries, including Romania. Despite progress, there remains a significant reliance on fossil fuels in some countries. Economic technologies for green hydrogen production are explored, with a note that its production alone does not solve all issues due to complex and costly compression and storage operations. The concept of impure green hydrogen, derived from biomass gasification, pyrolysis, fermentation, and wastewater purification, is also discussed. Economic efficiency and future trends in green hydrogen production are outlined. The article concludes with an analysis of hydrogen-methane mixture combustion technologies, offering a conceptual framework for economically utilizing green hydrogen in the transition to a green hydrogen economy.

Genome-wide identification and characterization of SNW/SKIP domain-containing proteins in plants.

Sessile organisms, such as plants, developed various ways to sense and respond to external and internal stimuli to maximize their fitness through evolutionary time. Transcripts and protein regulation are, among many, the main mechanisms that plants use to respond to environmental changes. SKIP protein is one such, presenting an SNKW interacting domain, which is highly conserved among eukaryotes, where SKI interacting protein acts in regulating key processes. In the present work, many bioinformatics tools, such as phylogenetic relationships, gene structure, physical-chemical properties, conserved motifs, prediction of regulatory cis-elements, chromosomal localization, and protein-protein interaction network, were used to better understand the genome-wide SNW/SKIP domain-containing proteins. In total, 28 proteins containing the SNW/SKIP domain were identified in different plant species, including plants of agronomic interest. Two main protein clusters were formed in phylogenetic analysis, and gene structure analysis revealed that, in general, the coding region had no introns. Also, expression of these genes is possibly induced by abiotic stress stimuli. Primary structure analysis of the proteins revealed the existence of an evolutionarily conserved functional unit. But physicochemical properties show that proteins containing the SNW/SKIP domain are commonly unstable under in vivo conditions. In addition, the protein network, demonstrated that SKIP homologues could act by modulating plant fitness through gene expression regulation at the transcriptional and post-transcriptional levels. This could be corroborated by the expression number of gene copies of SKIP proteins in many species, highlighting it's crucial role in plant development and tolerance through the course of evolution.

Potential Transcriptional Enhancers in Coronaviruses: From Infectious Bronchitis Virus to SARS-CoV-2.

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at the rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally defined coronaviral enhancers, we detected via an in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian infectious bronchitis virus (IBV) to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The duplex open state would pair with remote sequences in the viral genome and modulate the expression of downstream crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation, possibly driven by decreased open reading frame (ORF)3a immune evasion protein expression. If validated experimentally, the annotated enhancer sequences could inform structural prediction tools and antiviral interventions.

Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome

PurposeOral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. MethodsExome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. ResultsIn 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. ConclusionWe introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

Evolution from basic to advanced structure of fulvic acid and humic acid prepared by food waste

Fulvic acid (FA) and humic acid (HA) are common polyacids in nature. However, the evolutionary process of their basic and advanced structures is still unclear. FA and HA were separated into five molecular weight components to investigate the process of evolution from small to large molecules. The primary structure analysis showed that FA were rich in CN, COOH and OH content, while HA were rich in (CH2)n, NH2 and CC. Moreover, with the molecular weight increasing, the structures could complement each other to maintain the hydrophilic or hydrophobic balance. The 2D-COS spectroscopy demonstrated that during the growth of FA, COOH, NH2 and OH firstly respond. On the other hand, during the growth of HA, NH2 and (CH2)n firstly respond. In addition, advanced structure of FA was affected by intramolecular hydrogen bonds and π − π interaction. HA was affected by hydrophobic interactions due to the abundance of hydrophobic groups, primarily (CH2)n and benzene rings. 3D conformational fitting and particle size characterization confirmed that the interaction forces determine that FA and HA become tightly and loosely molecules respectively. This study is to further explore the geochemical formation and evolution process of FA and HA molecules.

In silico analysis of a novel hypothetical protein (YP_498675.1) from Staphylococcus aureus unravels the protein of tryptophan synthase beta superfamily (Try-synth-beta_ II)

BackgroundStaphylococcus aureus is a gram-positive spherical bacteria and the most common cause of nosocomial infections in the world. Given its clinical significance, the genome sequence of S. aureus has been elucidated to enhance our comprehension of its lifestyle and pathogenicity. The research aimed to summarize a potential hypothetical protein that may play an important role in S. aureus virulence and pathogenicity, covering its anticipated structure, probable biological functions, and importance in this context. ResultsA hypothetical protein, YP_498675.1 with 281 amino acid residues of S. aureus, was chosen for analysis and modeling by several bioinformatics tools and databases in this work. According to primary and secondary structure analyses, YP_498675.1 is a stable hydrophilic protein with a significant proportion of α–helices. Subcellular localization predictions by CELLO, PSORTb, and SOSUI server indicate that it is a cytoplasmic protein. NCBI-CDD, Pfam, and InterProScan functional genomics research revealed that the hypothetical protein may include the pyridoxal phosphate (PLP)-dependent 2, 3-diaminopropionate biosynthesis protein SbnA domain. In the homology modeling method, the HHpred server was employed to create its 3D structure using the template structure of a Staphyloferrin B precursor biosynthetic enzyme SbnA bound to PLP (PDB ID: 5D84_A), an X-ray diffraction model having 100% sequence identity with the hypothetical protein. After energy minimization, several quality assessments and validation factors determined that the generated protein model was reliable and of reasonable quality. ConclusionThe present study has characterized and functionally annotated the hypothetical protein YP_498675.1 of S. aureus. Further experimental validation would aid in determining the actual function of YP_498675.1 as well as confirm the protein’s value as a therapeutic target.

Molecular Modelling and Structure Analysis of Lectins from Vigna linearis

A study was conducted during the year 2022–2023 for in silico analysis of two Vigna linearis lectins in the computer lab, CDFT, Jodhpur, Rajasthan, India. Amino acid sequences of both lectins were obtained from NCBI database and analyzed using different computational tools. Primary structure analysis using ProtParam server revealed acidic, stable and hydrophobic nature of both VLL proteins. Both proteins were predicted heavily glycosylated with a single signal peptide cleavage site (A26-A27). High sequence similarity of both VLL proteins was observed with adzuki, rice bean and moth bean lectins. Both VLL proteins had high proportions of β-sheets in their secondary structure. Good quality 3D structures for both VLL proteins were modelled using Modeller software. A Jelly roll fold, also known as β-sandwich structure was identified in the 3D structure of both VLL proteins. Profunc server annotated both lectins as a carbohydrate binding regulatory protein with significant role in plant defense (GO terms: 0065007, 0006952, 0050896, 0006950, 0009607, 0030246). Galaxy Site web server predicted galactose, A2G (2-acetamido-2-deoxy-α-D-galactopyranose), MFU (Methyl α-L-fucopyranoside) and adenine as common binding ligands for both VLL proteins. Lig Plot analysis revealed hydrogen bonding and hydrophobic interactions were the major bonding interactions between VLL proteins and putative ligands. The results of the study would provide a base for conducting future research on Vigna linearis lectins for different applications.

Towards sustainability in post-disaster constructions with a modular prefabricated structure

ABSTRACT In response to the challenges faced by post-disaster construction, the present study endeavours to offer an improved solution in a practical manner. To this end, a critical analysis of modular systems that can be employed in post-disaster construction is conducted, which is succeeded by the set of required features. Considering these features, a system based on modular prefabricated components (MPC) is presented, which effectively eliminates all hitches while demonstrating facilitated solutions. From a practical standpoint, a comprehensive description of the structure’s dimensions, assembly steps and design constraints are proffered, accompanied by primary structural analysis factoring in the highest risk categories. The applicability and limitations of the system in post-disaster scenarios are also discussed in comparison to other modular construction methods. The findings indicate that the system exhibits high levels of disjoint-ability and collectability, flexibility and customisability, developability and repairability, in addition to being cost- and time-efficient. Furthermore, it demonstrates a favourable response to the predicaments posed by post-disaster situations, such as logistics, construction process, safety, seismic behaviour and environmental effects. Overall, this research highlights the applicability and potential of MPC systems in post-disaster construction and delivers a valuable output for policymakers, managers, architects and engineers involved in this regard.

Diagenesis, compaction strain and deformation associated with chert and carbonate concretions in organic‐rich marl and phosphorite; Upper Cretaceous to Eocene, Jordan

ABSTRACTThis paper presents an integrated petrographic–geochemical–geomechanical study of the growth mechanisms of carbonate and chert concretions observed at outcrop and core from the Upper Cretaceous to Eocene organic‐rich carbonate mudrocks, central Jordan. It provides evidence for displacive and replacive concretion growth from the analysis of primary lithological characteristics, compaction strain and deformation structures associated with concretion growth. Concretions were analysed to determine the primary lithological controls on their development and the measurement of strain in the host rock to develop a method for constraining the growth mode and their paragenesis. Concretions exhibit either a replacive or displacive growth mode largely dependent on the original host lithology. Displacive concretions exhibit irregular shapes and semi‐fibrous internal structures in contrast to regular shapes and microcrystalline textures observed for replacive concretions. Cement fraction is high in both carbonate concretion types, indicating early formation in high‐porosity sediments at shallow burial depths. The strain field around displacive concretions is vertically asymmetrical. Conversely, it is symmetrical with uniform differential compaction for the replacive concretions. Evidence for displacive growth comes from triangular areas of chert at the lateral margins of some carbonate concretions, interpreted as areas of reduced strain. Another indicator is the forced asymmetrical folding of heterolithic host rocks around displacive concretions, with displacive carbonate units separated by trace laminae of the original (chert) beds. Enveloping chert beds exhibit early‐formed radial silica fractures with increased aperture size in the areas of maximum curvature. Carbon isotopic signatures of carbonate concretions show a strong correlation between concretion centres and host rock, suggesting a relatively shallow depth (first few tens of metres) of initial growth. Carbonate concretions are interpreted to have formed at shallow depths in the presence of alkaline pore waters rich in dissolved organic carbon in the presence of Mg2+ ions, available organic matter and redox‐sensitive metals such as U and Mo. A paragenetic history for the different concretion types is presented.

MODELLING PRECISION CHARACTERISATION OF FIMBRIAL PROTEIN (fimA) OF Edwardsiella tarda AND Edwardsiella piscicida

Edwardsiellosis caused by Edwardsiella tarda and Edwardsiella piscicida is one of the most common catfish diseases. For any bacterium to cause an infection, it has to adhere to and invade the host. Major fimbrial protein (fimA) plays an important role in the adherence of Edwarsiella spp. using an in-silico approach, physicochemical properties, secondary structure, functional properties and homology modelling of fimA protein from E. tarda and E. piscicida were analysed in this study. The primary structural analysis indicated that all proteins are soluble, stable and hydrophobic. Secondary structures analysed using the SOPMA tool revealed that extended strands, random coils and alpha helices are predominant amidst secondary structural elements. The homology models of these proteins were built using SWISS-MODEL and their qualities were validated using ProSA, ProQ, PROCHECK and RAMPAGE analysis. The validated models were verified as good structures. The results suggested that the fimA can be a potential target for diagnosis and drug development to combat edwardsiellosis infection.

Engineering mesophilic GH11 xylanase from Cellulomonas flavigena by rational design of N-terminus substitution.

Xylanase, a glycoside hydrolase, is widely used in the food, papermaking, and textile industries; however, most xylanases are inactive at high temperatures. In this study, a xylanase gene, CFXyl3, was cloned from Cellulomonas flavigena and expressed in Escherichia coli BL21 (DE3). To improve the thermostability of xylanase, four hybrid xylanases with enhanced thermostability (designated EcsXyl1-4) were engineered from CFXyl3, guided by primary and 3D structure analyses. The optimal temperature of CFXyl3 was improved by replacing its N-terminus with the corresponding area of SyXyn11P, a xylanase that belongs to the hyperthermostable GH11 family. The optimal temperatures of the hybrid xylanases EcsXyl1-4 were 60, 60, 65, and 85°C, respectively. The optimal temperature of EcsXyl4 was 30C higher than that of CFXyl3 (55°C) and its melting temperature was 34.5°C higher than that of CFXyl3. After the hydrolysis of beechwood xylan, the main hydrolysates were xylotetraose, xylotriose, and xylobiose; thus, these hybrid xylanases could be applied to prebiotic xylooligosaccharide manufacturing.

Molecular Evolutionary Relationships of Side-Necked Turtle Hemoglobin:cDNA-Derived Primary Structures and Computer-Assisted Tertiary Structuresof α<sup>A</sup>, α<sup>D</sup>, and β Globin Chains

Extant side-necked turtles consist of three families: Chelidae, Pelomedusidae, and Podocnemididae. The lasttwo families (the subject of this study) include five genera, three of which are endemic to Africa (Pelomedusa andPelusios) and Madagascar (Erymnochelys), while the other two (Podocnemis and Peltocephalus) are restrictedto South America. This study was undertaken to determine the molecular relationships of side-necked turtlesbased on the primary and tertiary structures of α^A, α^D, and β globin chains as deduced by cDNA sequencing and amachine-learning algorithm for protein structure prediction, AlphaFold2. Analyses of the globin primary structures of four species from different genera provide three major insights. (1) The genera Pelomedusa and Pelusiosmight be endemic taxa that diversified in Africa after the Gondwana separation. (2) Although the extant species ofErymnochelys and Podocnemis are restricted to Africa and South America, respectively, they are closely related toeach other, and thus, these genera apparently diversified during the breakup of Gondwana. Thus, the colonizationof the ancestral population of Podocnemis unifilis in South America might have coincided with continental drift.(3) The divergence times of the four species based on globin genealogy were estimated to have begun 120–11million years ago (mya), which generally coincides with the break-up of Gondwana 135–65 mya. In addition,tertiary structures predicted by AlphaFold2 were compared using a PyMOL overlaying technique. An in-depth examination of the four α^A globin chains revealed considerable consistency among species, and a similar pattern wasobserved for α^D and β globin chains. Additionally, species-specific differences were recognized among the primarystructures, and their higher-order structures (i.e., tertiary structures) were consistent, offering new information onthe molecular phylogeny of side-necked turtles.

Isolation, Computational Characterization and Structure Prediction of Chitinase Gene-Bbchit of Beauveria bassiana

Beauveria bassiana is an entomopathogenic fungus that is used as a biopesticide for the control of many insect pests in agriculturally important crops. B. bassiana produces many extracellular hydrolytic enzymes that degrade the insect cuticle. Among them, the fungal exochitinase mainly assists in the infection by degrading the chitin present in the insect cuticle. To characterize the exochitinase from B. bassiana, the chitinase gene (Bbchit) of 1050bp was amplified from the genomic DNA through PCR using gene-specific primers. This chitinase gene was cloned into the pJET1.2 cloning vector, confirmed through sequencing. The sequence (GI; OP114061) has been deposited in the NCBI database and used for bioinformatics analysis. The various Physico-chemical properties of chitinase protein such as pI, EC, AI, GRAVY and instability index were predicted using primary structure analysis. Using MEGA11 software, the translated chitinase protein- Bbchit showed that it closely resembles with the QJE37897.1 (chitinase Chit37). Primary and secondary structure analysis helps to characterize the functionality of chitinase protein (Bbchit). The 3D structure of B. bassiana was predicted using Robetta, an abinitio prediction method and confirmed through in-silico using the SAVESv6.0 (PROCHECK) server. The isolated exochitinase gene from B. bassiana has been characterized for use as a bio-control agent for the control of insect pests in crop plants.

Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase.

Copper radical oxidases (CROs) from Auxiliary Activity Family 5, Subfamily 2 (AA5_2), are organic cofactor-free biocatalysts for the selective oxidation of alcohols to the corresponding aldehydes. AA5_2 CROs comprise canonical galactose-6-oxidases as well as the more recently discovered general alcohol oxidases and aryl alcohol oxidases. Guided by primary and tertiary protein structural analyses, we targeted a distinct extended loop in the active site of a Colletotrichum graminicola aryl alcohol oxidase (CgrAAO) to explore its effect on catalysis in the broader context of AA5_2. Deletion of this loop, which is bracketed by a conserved disulfide bridge, significantly reduced the inherent activity of the enzyme toward extended galacto-oligosaccharides, as anticipated from molecular modeling. Unexpectedly, kinetic and product analysis on a range of monosaccharides and disaccharides revealed that an altered carbohydrate specificity in CgrAAO-Δloop was accompanied by a complete change in regiospecificity from C-6 to C-1 oxidation, thereby generating aldonic acids. C-1 regiospecificity is unprecedented in AA5 enzymes and is classically associated with flavin-dependent carbohydrate oxidases of Auxiliary Activity Family 3. Thus, this work further highlights the catalytic adaptability of the unique mononuclear copper radical active site and provides a basis for the design of improved biocatalysts for diverse potential applications.

Selenium-containing polysaccharides isolated from Rosa laevigata Michx fruits exhibit excellent anti-oxidant and neuroprotective activity in vitro

Selenium-containing polysaccharides have potential as an organic selenium dietary supplement, owing to their low toxicity, few side effects, and easy absorption attributes. In this study, we isolated two novel homogeneous selenium-containing polysaccharides from Rosa laevigata Michx fruits (Se-RLFPs). Results from primary structural analysis revealed that Se-RLFPs were α – pyranose, and were both composed of rhamnose, xylose, glucose with an average molecular weight of 24 and 16 KDa, respectively. Selenium contents in Se-RLFP-I and Se-RLFP-II were 16.49 μg/g and 21.61 μg/g, respectively. Results from analysis of antioxidant and neuroprotective activity of the polysaccharides revealed that Se-RLFPs had a radical scavenging effect. Specifically, they effectively protected SH-SY5Y cells from H2O2-induced damage by enhancing antioxidant enzyme activities (SOD), total antioxidant capacity (T-AOC) and suppressing malondialdehyde (MDA) levels. Western blots showed that the underlying mechanisms of action may be related to the Nrf2/HO-1 signaling pathway. Taken together, these results suggested that Se-RLFPs have potential as a pharmaceutical agent for treatment of neurodegenerative diseases (NDDs) or as a selenium-complementary ingredient in functional foods.

Continuous pedagogical education: value orientations of entry into profession

Introduction. The topic of this article is relevant because there is no consistency in the formation of professional pedagogical values and orientations in the implementation of educational programs at different stages of teacher training. The purpose of the study is a comparative analysis of the value-semantic orientations of professional pedagogical activity of future teachers at the stage of training in a pedagogical college and university. Methods. The study involved students of pedagogical colleges (N=147) of the Yaroslavl region and students of the Yaroslavl State Pedagogical University named after K.D. Ushinsky (N=253). To study professional values and meanings, a questionnaire was developed that includes three lists of instrumental values focused on the child, environment and profession and a set of Must-topics that reveal the respondents' attitude to the priorities and problems of teacher training. Empirical data were processed using the methods of primary descriptive statistics, correlation, structural, and factor analysis. The results of the study. Significant differences in the value-semantic structure of the profession of future teachers were revealed: for university students, the enrichment of students' life experience is more significant than for college students (N=7,061, p<0,05); university students value knowledge of technologies of diagnostic and correctional-developmental work with students (N=8,203, p<0,05) and ways of developing students' self-management (N=7,749, p<0.05) more. Structural analysis revealed the first place of student development values in professional activity (average values of 20,4 for college students and 15,8 for university students). Factor analysis showed a high value of knowledge values for college and university students. At the same time, college students have more factor weight of knowledge of ways to cooperate with all participants in the educational process (0,609), and university students have more factor weight of knowledge of ways to involve students in various activities (0,565). Conclusion. The conducted research has shown that students' value orientations are equal to the guidelines declared by the pedagogical community. The hypothesis about the differences in the value-semantic orientations of future teachers at various stages of professional training has been confirmed. Students of a pedagogical university have a more balanced structure of values-goals: both in the value system and in individual groups of instrumental values, the priority sequence "child-environment-profession" is visible. College and university students attach more importance to values-knowledge. It is necessary to increase practical training at all stages of teacher training.

Structural basis for the recognition of methylated histone H3 by the Arabidopsis LHP1 chromodomain

Arabidopsis LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), a unique homolog of HP1 in Drosophila, plays important roles in plant development, growth, and architecture. In contrast to specific binding of the HP1 chromodomain to methylated H3K9 histone tails, the chromodomain of LHP1 has been shown to bind to both methylated H3K9 and H3K27 histone tails, and LHP1 carries out its function mainly via its interaction with these two epigenetic marks. However, the molecular mechanism for the recognition of methylated histone H3K9/27 by the LHP1 chromodomain is still unknown. In this study, we characterized the binding ability of LHP1 to histone H3K9 and H3K27 peptides and found that the chromodomain of LHP1 binds to histone H3K9me2/3 and H3K27me2/3 peptides with comparable affinities, although it exhibited no binding or weak binding to unmodified or monomethylated H3K9/K27 peptides. Our crystal structures of the LHP1 chromodomain in peptide-free and peptide-bound forms coupled with mutagenesis studies reveal that the chromodomain of LHP1 bears a slightly different chromodomain architecture and recognizes methylated H3K9 and H3K27 peptides via a hydrophobic clasp, similar to the chromodomains of human Polycomb proteins, which could not be explained only based on primary structure analysis. Our binding and structural studies of the LHP1 chromodomain illuminate a conserved ligand interaction mode between chromodomains of both animals and plants, and shed light on further functional study of the LHP1 protein.

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.